M. Rafiq Islam

Active Site Residues of Human β-Glucuronidase EVIDENCE FOR GLU540 AS THE NUCLEOPHILE AND GLU451 AS THE ACID-BASE RESIDUE

Human β-glucuronidase (hGUSB) is a member of family 2 glycosylhydrolases that cleaves β-d-glucuronic acid residues from the nonreducing termini of glycosaminoglycans. Amino acid sequence and structural homology of hGUSB and Escherichia coliβ-galactosidase active sites led us to propose that residues Glu451, Glu540, and Tyr504 in hGUSB are involved in catalysis, Glu451 being the acid-base residue and Glu540 the nucleophile. To test this hypothesis, we introduced mutations in these residues and determined their effects on enzymes expressed in COS cells and GUSB-deficient fibroblasts. The extremely low activity in cells expressing Glu451, Glu540, and Tyr504 hGUSBs supported their roles in catalysis. For kinetic analysis, wild type and mutant enzymes were produced in baculovirus and purified to homogeneity by affinity chromatography. Thek cat/K m values (mm −1·s−1) of the E540A, E451A, and Y504A enzymes were 34,000-, 9100-, and 830-fold lower than that of wild type hGUSB, respectively. High concentrations of azide stimulated the activity of the E451A mutant enzyme, supporting the role of Glu451 as the acid-base catalyst. We conclude that, like their homologues in E. coli β-galactosidase, Glu540 is the nucleophilic residue, Glu451 the acid-base catalyst, and Tyr504 is also important for catalysis, although its role is unclear. All three residues are located in the active site cavity previously determined by structural analysis of hGUSB.

MAP/ERK kinase kinase 1 (Mekk1) mediates transcriptional repression by interacting with polycystic kidney disease-1 (PKD1) promoter-bound p53 tumor suppressor protein

Mitogen-activated protein kinase (MAPK) cascades regulate a wide variety of cellular processes that ultimately depend on changes in gene expression. We have found a novel mechanism whereby one of the key MAP3 kinases, Mekk1, regulates transcriptional activity through an interaction with p53. The tumor suppressor protein p53 down-regulates a number of genes, including the gene most frequently mutated in autosomal dominant polycystic kidney disease (PKD1). We have discovered that Mekk1 translocates to the nucleus and acts as a co-repressor with p53 to down-regulate PKD1 transcriptional activity. This repression does not require Mekk1 kinase activity, excluding the need for an Mekk1 phosphorylation cascade. However, this PKD1 repression can also be induced by the stress-pathway stimuli, including TNFα, suggesting that Mekk1 activation induces both JNK-dependent and JNK-independent pathways that target the PKD1 gene. An Mekk1-p53 interaction at the PKD1 promoter suggests a new mechanism by which abnormally elevated stress-pathway stimuli might directly down-regulate the PKD1 gene, possibly causing haploinsufficiency and cyst formation.

Retinoic acid-dependent activation of the polycystic kidney disease-1 (PKD1) promoter

The retinoic acids all-trans retinoic acid (AT-RA) and 9-cis retinoic acid (9C-RA) and the retinoic acid receptors RAR and RXR significantly induce transcriptional activity from a 200-bp PKD1 proximal promoter in transfected mammalian cells. This PKD1 promoter region contains Ets, p53, and GC box motifs, but lacks a canonical RAR/RXR motif. Mutagenesis of the Ets sites did not affect RA induction. In contrast, GC box mutations completely blocked stimulation by AT-RA and by RXRbeta or RARbeta. Mithramycin A, which prevents Sp1 binding, significantly reduced basal promoter activity and suppressed upregulation by AT-RA and RXR. The 200-bp proximal promoter could not be induced by AT-RA in Drosophila SL2 cells, which lack Sp1, but could be activated in these cells transfected with exogenous Sp1. Small interfering RNA knockdown of Sp1 in mammalian cells completely blocked RXRbeta upregulation of the promoter. These data indicate that induction of the PKD1 promoter by retinoic acid is mediated through Sp1 elements. RT-PCR showed that AT-RA treatment of HEK293T cells increased the levels of endogenous PKD1 RNA, and chromatin immunoprecipitation showed the presence of both RXR and Sp1 at the PKD1 proximal promoter. These results suggest that retinoids and their receptors may play a role in PKD1 gene regulation.

Hepcidin Regulation by Bone Morphogenetic Protein Signaling and Iron Homeostasis

Homeostasis Marianna Rodova1, Seunghwan Kim1, M. Abdul Mottaleb2 and M. Rafiq Islam1* 1Biochemistry Laboratory, Northwest Missouri State University, Maryville, Missouri, USA 2Center for Innovation and Entrepreneurship, Northwest Missouri State University, Maryville, Missouri, USA *Corresponding author: Rafiq Islam, Biochemistry Laboratory, Northwest Missouri State University, Maryville, USA, Tel: 660-562-3118; E-mail: